Degenerative diseases are a major health issue of the twenty-first century, largely due to the global increase in the median age of humans. Recent findings suggest, however, that the human body retains a capacity for tissue specific stem cell activity even in regions formerly thought to be completely quiescent, such as the brain. Because the ability to induce and regulate cell type specific regeneration programs would represent the ultimate solution to degenerative diseases and conditions, a high-throughput vertebrate model system capable of fully elucidating the genetics and pharmacology of cellular regeneration is needed.
Zebrafish are an established model organism for investigating the genetics and pharmacology of vertebrate biology: Zebrafish are economical to maintain in the laboratory environment and are highly fecund; a single female is capable of generating hundreds of offspring per week. The zebrafish embryo develops externally and is transparent, allowing direct visualization of cellular and tissue developmental processes as they proceed in vivo, thereby facilitating large-scale genetic and small molecule drug screens. In the past several years numerous publications have reported transgenic fish lines expressing green fluorescent protein (GFP) in cell-type restricted expression patterns (Gong et al., 2001; Kennedy et al., 2001; Long et al., 1997; Moss et al., 1996; Motoike et al., 2000; Park et al., 2000). To date, studies using fluorescent transgenic zebrafish have focused mainly on imaging cells and tissues as they develop. Such transgenic zebrafish lines—in addition to promoting developmental investigations of tissue morphogenesis—facilitate genetic and pharmacological screens by allowing high-resolution imaging of discrete cell populations.
Moreover, as a disease model system, transgenic zebrafish provide a unique opportunity to elucidate cellular regeneration at the level of the entire genome of a vertebrate organism. This is due to a confluence of the required factors in this organism: 1) A robust capacity for cellular regeneration in a vertebrate; 2) Amenability to a forward genetics approach of random mutagenesis based screening; 3) Transparency, during embryonic, larval, and even into adult stages (given the proper genetic background) which allows the process of regeneration to be directly observed over time in the living organism, and; 4) Amenability to high-throughput genetic and pharmacological screening. Compared to other genetic models, zebrafish have the advantage of being more akin to humans than yeast, worms, or flies in terms of body plan (vertebrate) and genetic homology (75% and greater similarity to humans) and in being far more economical than mice. These are just a few of the reasons that zebrafish have emerged as the leading vertebrate model organism for large-scale ‘forward genetics’ based mutational screens (Driever et al., 1996; Haffter et al., 1996; Henion et al, 1996; Mullins et al., 1994). Furthermore, zebrafish have a remarkable regenerative capacity that extends even to their nervous system (Poss et al., 2003; Zupanc, 2001).
Pro-drug conversion systems have been reported by researchers as a method for targeted ablation of cancer cells (Denny, 2001). Several methods of delivering pro-drug converting systems specifically to cancer cells have been have been developed including, virus-directed enzyme pro-drug therapy (VDEPT), antibody-directed enzyme pro-drug therapy (ADEPT), and gene-directed enzyme pro-drug therapy (GDEPT). This system can also be applied to targeted and regional elimination (“ablation”) of normal cells in order to study regeneration. Of particular interest are well studied pro-drug converting enzymes, such as bacterial nitroreductase, for which numerous pro-drugs with specific properties have been defined. For instance, certain pro-drugs can be used for targeted cell-specific ablation while other drugs promote more widespread regional ablation—whereby cells in the general vicinity of nitroreductase-expressing cells are also eliminated (Bridgewater et al., 1997). The regional ablation protocol, also called the ‘bystander effect’, can be used to model injury paradigms. In addition transgenic mice expressing prodrug conversion enzymes are able to specifically ablate cells in which the enzyme is expressed when these mice are treated with the appropriate prodrug (Felmer et al., 2002; Isles et al., 2001; Ma et al., 2002). Finally, a fusion protein between GFP and nitroreductase has been described which retains the function of both components in cell culture (Medico et al., 2001). Such fusion proteins ensure that the ablation component and reporter component do not segregate away from each other and allow definitive detection of all ablation competent expressing cells and regions.
Regenerative therapies are highly desired as an approach to curing degenerative conditions. Degenerative conditions include disorders such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, spinal cord injury, traumatic brain injury, multiple sclerosis, cerebral palsy, osteoarthritis, and other age related forms of degeneration. Despite generally useful therapies including medicinal therapies currently available to ameliorate the symptoms of these afflictions, there is a substantial need for improved research tools to identify new compounds and to establish enhanced therapies for the treatment of these and other degenerative ailments.
Technical Problem: Ablation technology must fulfill several requirements in order to take advantage of the inherent regenerative capacity of the zebrafish and suitability to high-throughput analysis for finding both genes and compounds useful for the treatment of degenerative disorders. These requirements include: 1) cell or tissue type specificity, 2) temporal control of ablation 3) adaptability to large scale high throughput analysis, and 4) ease of detection of both ablation and regeneration. A combination of all of these requirements is not available in current technology.